Despite numerous advances in cell culture procedures, the culture of differentiated epithelial cells, particularly from normal tissues, has remained especially difficult. It has been proposed that the shortcomings of cell culture techniques are primarily caused by cells being isolated from the extracellular matrix and from association with other cell types with which they may be interdependent. Culture methods, such as organ culture or the culture of tissue fragments, which retain tissues architecture, preserve the differentiative state of the cells, whereas clonal cell cultures usually undergo a dedifferentiative process. Thus, to culture differentiated cells it is necessary to ascertain the critical variables of the tissue matrix and to evolve culture procedures dependent upon them, something the prior art had failed to do.
In the aforementioned patent application, Ser. No. 089,167, now U.S. Pat. No. 4,352,887, connective tissue fibers or biomatrix were disclosed and claimed which, when used as a culture substrate, would simulate some of the cell-cell relationships of the tissue matrix relevant to differentiated cells. On substrates of reconstituted basement membrane and in medium supplemented with hormones, serum, and with conditioned medium from feeder layers of primary cultures of fibroblasts these differentiated cells could be cultured.
The prior art, however, with regard to cell culture substrates and isolation of connective tissue fibers respectively produced either pure collagen (typically of only one type) or produced biomatrix which would be highly toxic to cells if used in culture.
For instance, the reference by Price in Chemical Abstracts, entitled "Preparation and use of Rat Tail Collagen" 84: 71179K, 194 (1976) discloses the preparation of a cell culture substrate which comprises only Type I collagen. This is typical of most prior art references for preparation of cell culture substrates. Significantly, the only components of connective tissue-derived fibers studied to date by the prior art have been those which were soluble in one solution or another, because of the prior art's use of a positive selection system (the solubilization of the desired component and subsequent purification of it as an isolated individual entity). The connective tissues-derived fibers isolated by the method claimed herein, however, is a mixture of components, only some of them being collagens. The relevance of this to the differentiative and/or proliferative capacity of cells is due to the synergistic interactions of all these components in their affects on cells. Although cells will respond to one component or another derived from the connective tissue-derived fibers, e.g., collagens, their more normal, physiological response is observed only when they are in contact with the complete connective tissue-derived fibers. Indeed, the collagen fibers isolated by the prior and the other components which make up the "connective tissue fibers", in part but not in toto, have long been known individually to enhance primary cells cultures of differentiated epithelial cells. The complete connective tissue-derived fibers isolated by the disclosed invention, however, have been found to be significantly superior than the individual components alone, since they are comprised of multiple components which are found to synergistically interact on a broader front to enhance cell culturing abilities. All cells are optimally differentiated when they are maintained in vivo. The biomatrix isolated by this invention, when used in culture, provides an in vitro simulation of those in vivo conditions.
The connective tissue-derived fibers also vary in chemical composition from tissue to tissue. Although there are the same types of components (collagens, non-collagenous proteins, and carbohydrates) in all tissues, the specific forms of collagen or other components is different in different tissues. For example:
Skin: Type I collagen predominates. This is typically the type of collagen isolated by the prior art for use in cultures. In association with Type I collagen is an anchorage protein, fibronectin, and proteoglycan, dermatan sulfate. There are other non-collagenous proteins also associated with the Type I collagen but they have not been chemically purified or characterized. PA0 Liver: Type IV collagen predominates. In association with Type IV collagen is an anchorage protein, laminin, and proteoglycan, heparan sulfate. Since Type IV collagen is distinguished from the other collagens by its large number of sulfhydryl groups and disulfide bonds, it is in association with many noncollagenous proteins (most of them glycoproteins or proteoglycans) which are bound to the collagen covalently through these sulfur linkages. Indeed, the mixture of specific proteins will be unique to each different type of tissue.
Bone: Type II collagen predominates. In association with Type II collagen is an anchorage protein, chondronectin and proteoglycan, chondroitin sulfate. As above there are other noncollagenous proteins and carbohydrates, undefined, which are found in association with Type II collagen.
Typically, the prior art references purify the collagen Type I due to its solubility in dilute acids) to various degrees and then either smear it on plates, gel it (non-fibrous; denatured collagen), or reconstitute it into fibers. Purified Type I collagen gels, coats, or fibers have long been used as collagenous substrates for primary cultures of normal epithelial cells. However, not all normal epithelial cells attach to Type I collagen and furthermore the connective tissue-derived fibers isolated by the disclosed invention contains not only Type I collagen but Type II, III and IV and many other components other than collagen. One cannot see the fully normal state of the cells without all of these components. This is because of synergistic interactions between these components and the cultured cells. This synergism is further enhanced by the effect of cell-specific hormones and conditioned medium. One cannot replace the complex composition found in the connective tissue-derived fibers with only one or another of the components present in those fibers.
The prior art has also disclosed the preparation of biomatrix for purposes other than cell culture purposes. See Meezan, et al., entitled "A Simple, Versatile, Nondisruptive Method For The Isolation of Morphologically and Chemically Pure Basement Membranes from Several Tissues", in Life Sciences, Vol. 17, pp. 1721-1732. However, because the Meezan end product biomatrix is not being used for cell culture purposes, but merely for chemical analysis purposes, compounds which are toxic to cells in culture are utilized in the isolation and purification steps and are retained by the biomatrix. Specifically, the Meezan process utilizes a sodium azide solution at various steps and residual sodium azide is retained by the biomatrix. Sodium azide has been shown in the prior art to be toxic and mutagenic to mammalian cells in culture. See Seamenova, et al, "The Effects of Sodium Azide on Mammalian Cells Cultivated In Vitro", Mutation Research, Volume 71, pp. 253-261 (1980) and Jones, et al., "Toxicity and Mutagenicity of Sodium Azide in Mammalian Cell Cultures," Mutation Research, Volume, 77, pp. 293-299 (1980).
Furthermore, the Meezan process also utilizes a deoxycholate solution which also leaves a toxic residue in the end product biomatrix. Another significant distinction is that the Meezan reference does not involve the use of ribonuclease in addition to DNase. The use of ribonuclease and DNase enables the production of more pure connective tissue derived fibers, without any contaminating DNA or RNA, and this is essential for use as a tissue-specific cell culture substrate. The use of DNase in the Meezan article is merely to prevent "viscous gel"-like DNA from interfering with the handling of the biomatrix, but would not result in a similar functional product required when using connective tissue-derived fibers as a culture substrate.
It is therefore an object of this invention to provide a method for the preparation and isolation of a novel and non-toxic cell culture substrate.
It is another object of this invention to provide for the isolation of a biomatrix composition which, when utilized in cell cultures, provides an in vitro simulation of in vivo conditions.
The aforesaid objects as well as other objects and advantages will be made more apparent in reading the following description and the adjoined claims.